The genome is a virus'infectious material, thus proper packaging, storage, and disassembly are critical to successful infection. Yet, little is known about the three- dimensional structure of viral genomes. We developed a method for determining the structure of viral genomes that are packaged inside spherical (icosahedral) capsids. The technique uses images from cryogenic electron microscopy and allows three- dimensional reconstruction of the genome structure. The method works for simulated image data, and we propose to test the technique with images of satellite tobacco mosaic virus (STMV), a simple model system. Successful 3D reconstruction relies on averaging many particle images and the premise that the three-dimensional genome arrangement is consistent from particle-to-particle. We expect to extend our study to picornaviruses, a family of significant human pathogens, using poliovirus as our model system. We hypothesize that the genomes of individual viral species have a consistent 3D structure. By testing this hypothesis, we propose to test the feasibility of picornaviruses for our genome-reconstruction method. PUBLIC HEALTH RELEVANCE: For effective infection, a virus'genome must be arranged correctly in three dimensions. Understanding 3D genome architecture will give a fuller mechanistic picture of how viruses cause disease and may give insights into anti-viral therapies.